To assess the psychometric properties, including internal consistency, construct validity, criterion validity, criterion-group validity, and responsiveness, the Reviewed McGill Quality of Life Questionnaire (MQOL-R), into Brazilian Portuguese-(BrP). Also, to analyze the relationship of the BrP-MQOL-R with the scores on the Karnofsky Performance Scale (KPS) and on the Numerical Pain Scale (NPS 0–10).The BrP-MQOL-R was administered to a sample of 146 adults (men = 78). A team of experts translated the MQOL-R according to international guidelines. Convergent validity and Confirmatory factor analysis (CFA) was performed.The BrP-MQOL-R Cronbach’s alpha was 0.85. CFA supported the original four-factor structure, with the following revised model fit-indices: PCLOSE = 0.131, Tucker-Lewis Index (TLI) rho 2 = 0.918, incremental fit index (IFI) delta 2 = 0.936. The convergence validity is supported by a significant correlation between BrP-MQOL-R total scores and their subscales with KPS and with the single item related to the quality of life. And by a converse correlation with the pain scores in the NPS (0–10). Receiver operator characteristics (ROC) analysis showed subjects with KPS equal to or lower than 30% could be discriminated from those with scores on KPS higher than 30% by an area under the curve (AUC) = 0.71, sensitivity = 97%, and specificity = 92%).The BrP-MQOL-R proves to be a reliable instrument for assessing the quality of life (QOL) in palliative care (PC), with primary evidence of validity. BrP-MQOL-R presented adequate discriminate properties to identify distinct conditions that impact the QOL in PC. Palliative care is specialized medical care for people living with a severe illness with a life-threatening disease to prevent and relieve suffering through early identification, with appropriate evaluation and treatment of pain, physical, psychosocial, and spiritual problems. The palliative care provides relief from the symptoms to reduce the stress of the illness coming to the comfort of suffering and improvement of the Quality of Life (QOL) for both the patient and the family” . The QOLThe McGill Quality of Life Questionnaire-Reviewed , improveThus, we conduct the present study to examine the psychometric reliability of the translated MQOL-R for the Brazilian population . (I) We The protocol of this cross-sectional study was approved by the Ethics Committee Board of the Hospital de Clínicas de Porto Alegre, Brazil (protocol no 2019–0207). All subjects gave their written formal consent for participation or their caregivers. Figure Previously published guidelines carried out the procedures for the translation and cross-cultural adaptation of the MQOL-R to Brazilian Portuguese –13. We fTwenty patients assessed the comprehension of the item of BrP-MQOL-R. Among them, nine were inpatients, and 11 were women. The median age was 59.50 [interquartile ranges IQR) ] and the median of formal schooling was 8 years , respectively. They evaluated the meaning of the translated questions and the layout of the pre-final version of the BrP-MQOL-R and assessed each item's comprehension using a 10 cm visual analog scale . The median of comprehension of all items was 8.70 .A total of 157 patients over 18 years old at the Pain and Palliative Medicine Service of “Hospital de Clínicas de Porto Alegre, Brazil, from March 2019 to December 2019. Sixty-four in patients (43.8%) and 82 (56.2%) outpatients. Patients illiterate and those with cognitive impairment that prevented them from answering questions or communicating were excluded. Data were obtained by trained evaluators using a standardized questionnaire, the MQOL-R, the Performance de Karnofsky Scale, and a Numerical Pain Scale (NPS 0–10).NPS (0–10) was used to measure pain intensity, ranging from 0 (no pain) to 10 (the worst pain possible). They answered their pain level most of the time in the last 24 h and the pain score after taking pain medication.The Karnofsky Performance Status Scale (KPS) was used to quantifying functional status. The KPS is an 11-point rating scale that ranges from normal functioning (100) to dead (0). The KPS of < 30 the patients unable to perform these activities with or without assistance .The McGill Quality of Life Questionnaire-Revised (MQOL-R) consists of 14 items divided into four domains: The Single-Item Scale (SIS), physical symptoms (three items), feelings and thoughts (seven items), and social (three items). The overall scale has good internal reliability (α = 0.94) .We conducted descriptive statistics to examine the underlying assumptions of normality for all variables of interest. The Cronbach’s alpha and Spearman-Brown were used for the assessment of the MQOL-R’s reliability. For the BrP-MQOL-R, the maximum likelihood factor analyses with oblique rotation were conducted. We checked the scale's internal structure using confirmatory factor analysis (CFA) and establishing its reliability and validity. Items with a loading equal to or higher than 0.4 were retained to be considered relevant . FactorsThe demographic data and clinical characteristics are presented in Table The BrP-MQOL-R final 14-item had a satisfactory internal consistency (α = 0.85). The mean (SD) for all items of the scale was 6.09 .The MQOL-R scale and subscales and the total result were scored by averaging across items. We checked whether the findings in subscales differ from one another; we conducted a one-way repeated-measures ANOVA. The data comply with the variance sphericity . This result indicates that the results in the MQOL-R sub-scales differ from one another. The multiple comparison test by Bonferroni revealed that QOL in the subscale social [mean (standard deviation)] was highest in our sample , followed by existential [6.36 (2.10) P < 0.001 for all comparisons], psychological [5.21 (2.60)], and physical [4.88 (2.01)]. Other comparisons returned no significant difference.2 test  suggests insufficient fit, although this statistical tool is too restrictive and often points to rejecting a model with high samples involved. The chi-square/degree of freedom (CMIN/df = 1.608) reached a satisfactory value under 5. Following the strategy of presenting fit indices suggested by Hu and Bentler [We tested the internal structure of the MQOL-R using CFA, using the generalized least squares method. CFA revealed that all items were related to four specified factors, verifying the item's relationships and latent factors. Figure  Bentler  if the rThe correlation between the BrP-MQOL-R total scale and subscale scores is displayed in Table The pain scores on NPS (0–10) in two conditions, the pain level on most of the time in the last 24 h and relive of pain score when you take pain medication. The mean (SD) on the KPS was 59.18 (21.38). The mean score (SD) in the SIS measuring overall quality of life was 6.5 (2.54). The mean (SD) on the question of their pain level on most of the time in the last 24 h was 4.50 (3.72), and after taking pain medication was 2.06 (2.78).The responsiveness of the BrP-MQOL-R can be seen by the mean (standard deviation) of the total score. Patients with KPS equal to or lower than 30% could be discriminated from those with scores on KPS higher than 30%; the score on the BrP-MQOL-R was 4.83 (1.77) vs. 6.36 (1.95) (P = 0.00), respectively. Also, the scores of the QOL scale and subscales tend to be higher in subjects with the best functional status. That is means that this tool has properties to capture differences between patients in palliative care with the worst performance of those who have better functional status. We assessed the criterion validity by the screening accuracy to discriminate patients with KPS equal to or lower than 30% (n = 25) those with scores on KPS higher than 30% (n = 121) by non-parametric receiver operating characteristics (ROC) analyses an area under the curve (AUC) = 0.71, sensitivity = 97% and specificity = 92%).A regression analysis was used to assess if sex, hospitalization, formal education, and age could influence the score in the BrP-MQOL-R. The variables retained in the model were sex and the hospitalization at the time of assessment, the beta-coefficient was − 0.86  and 0.78 , respectively. That is, females and, if they were at home at the time of the assessment, showed higher scores.2 adjusted = 0.36; β = 0.49, t = 4.50, p < 0.001) than those found in the MQOL-R subscales (R2 adjusted = 0.36). A combination of two subscales was significant in predicting the SIS: Physical  and Existential .Separate regression analyses were performed to determine the global BrP-MQOL-R score and a combination of the BrP-MQOL-R subscales to predict the SIS. These models were adjusted by hospitalization at the time of assessment adjusted and sex. The total score predicted similar variance in the SIS  validated for Brazilian Portuguese


Solitary fibrous tumors (SFTs) are rare and can be misdiagnosed because of their various radiological appearances.To clarify the characteristic MRI findings of SFTs by analyzing their radiological-pathological correlation.Nine consecutive patients with SFT who underwent magnetic resonance imaging (MRI) prior to surgery were analyzed. Eight patients underwent contrast-enhanced MRI, and three underwent dynamic MRI. Radiological-pathological correlation analysis, co-occurrence matrix, run-length matrix, and histogram analysis were performed to assess the relationship between pathological findings T1- and T2-weighted images (T1-WI and T2-WI).ρ = –0.97, p < 0.0001) and endothelial cells .All nine lesions ranged in size from 20 to 36 mm. Seven lesions were located in the superior portion of the retrobulbar space found outside of the muscle cone, and two lesions in the inferior portion were located within it. No significant correlation was observed between the amount of collagenous tissue and the qualitative evaluation of the signal on T1-WI and T2-WI. Kurtosis on T2-WI was significantly correlated with the amount of collagenous tissue (Kurtosis in the histogram analysis on T2WI showed a strong correlation with the amount of collagenous tissue. Solitary fibrous tumors (SFTs) are rare spindle-cell neoplasms originating from the mesenchymal tissue. SFTs were initially thought to occur exclusively in the intrathoracic region; however, they have recently been reported to occur in extra-pleural sites, including the orbit . Althoug4This retrospective study was conducted in accordance with the World Medical Association Declaration of Helsinki and was approved by the institutional review board. Informed consent was waived.The data of subjects were collected from a pathology database, using the key words “orbit” and “solitary fibrous tumor” within the previous 10 years. All SFTs were resected and confirmed pathologically. Extra orbital tumors were excluded from the study. Among the patients with SFTs, MRI was performed on nine patients, who were subsequently included in this study. The nine patients comprised three men and six women, ranging in age from 14 to 77 years (median: 49 years).MRI was performed with a 1.5-Tesla (T) scanner . The MRI analysis included T1-weighted (T1-WI), T2-weighted (T2-WI), and diffusion-weighted imaging. Contrast-enhanced MRI was performed in 8 patients, and dynamic MRI was performed in three patients. T1-WI was performed under the following parameters: Repetition time (TR): 450 msec, Echo time (TE): 12 msec, Bandwidth (BW): 100 Hz, Flip angle (FA): 90°, matrix: 204 × 256 and Field of view (FOV): 150 × 150 m. A T2-WI with fat suppression was performed under the following parameters: TR: 3500 msec, TE: 96 msec, BW: 65 Hz, FA: 170°, matrix: 234 × 320 and FOV: 150 × 150 mm. A diffusion-weighted image was performed under the following parameters: TR: 4500 msec, TE: 71 msec, BW: 100 Hz, FA: 180°, matrix: 166 × 166 and FOV: 280 × 280 mm. A contrast-enhanced T1-WI was performed under the following parameters: TR: 650 msec, TE: 12 msec, BW: 120 Hz, FA: 90°, matrix: 204 × 256 and FOV: 150 × 150 mm.Two experienced radiologists (with 8 and 28 years of experience) performed the consensus readings. The following characteristics were evaluated from the MRI: Contour, location, and signal intensity on T1-WI or T2-WI, linear or curvilinear hypointense line in the lesion, and enhancement pattern. Signal intensities were determined by comparing them with the signal intensity of the cerebral cortex. Contours were classified as either well-defined or irregular. Location was classified as either above or below the center, and as either inside or outside of the muscle cone. Signal intensities on T1-WI and T2-WI were classified into 5 categories. Hypointensity was categorized as 1, isointensity was categorized as 3, and hyperintensity was categorized as 5. Category 2 was defined as samples showing a combination of hypointensity and isointensity. Category 4 was defined as samples showing mixed isointensity and hyperintensity. The curvilinear hypointense line was evaluated and classified as either present or absent. The enhancement pattern in the equilibrium phase was classified as either homogeneous or heterogeneous enhancement.Radiologists and radiological technologists delineated the region of interest on T1-WI and T2-WI. The ROIs were set at the entire tumor through all slices on T1-WI and T2-WI. The data acquired from each slice were summated to derive pixel-by-pixel images. Three different texture features  were extracted on a program developed using MATLAB after saving DICOM data into local storage.Pathological evaluations were performed by an experienced pathologist (more than 20 years of experience). Azan and Masson trichrome staining methods were used to observe the proliferation of collagenous fibers or fibrous components  and the Two radiologists and one pathologist discussed the findings and performed radiological-pathological correlation analyses between areas of interest.p-value of less than 0.05 was considered to indicate a statistically significant difference.Data are expressed as mean ± standard deviation. The radiological-pathological correlation was estimated. Spearman’s rank correlation coefficient was performed to compare MRI findings and texture analysis with pathological findings. A All nine lesions ranged in size from 20 to 36 mm (median: 28 mm).Table 1. The contour of the lesion was well-defined in six lesions and irregular in three lesions. Regarding the location, seven lesions were found to be in the superior portion, and two lesions were in the inferior portion. Seven lesions were inside the muscle cone, and two lesions were outside the muscle cone. On T1-WI, homogeneous isointensity was displayed in four lesions, heterogeneous isointensity and hypointensity in two lesions, and heterogeneous isointensity and hyperintensity in three lesions. On T2-WI, marked homogeneous hyperintensity was displayed in one lesion, heterogeneous isointensity and hyperintensity in four lesions, homogeneous isointensity in one lesion, heterogeneous isointensity and hypointensity in two lesions and marked hypointensity in one lesion. A linear or curvilinear hypointense line was observed in six lesions and was absent in three lesions. A homogeneous enhancement pattern in the equilibrium phase was displayed in six lesions and a heterogeneous enhancement in two lesions.The classified categories of MRI findings are summarized in ρ = 0.262, p = 0.49) and T2-WI  . A significant correlation was not observed between the level of CD31 staining and homogeneity of the enhancement pattern  .There was no significant correlation between the amount of collagenous tissue and signal intensity on both T1-WI  and was strongly correlated with the amount of collagenous tissue . The other parameters were not significantly correlated with the pathological findings.The summary of three texture analyses  is shown in The qualitative signal intensity on T2WI and the amount of collagenous tissue confirmed pathologically showed no significant correlation. However, the kurtosis in the histogram analysis showed a strong correlation with the amount of collagenous tissue. This result indicates that the texture analysis may allow the characterization of SFTs, which is difficult by visual inspection. On the other hand, high-level texture analyses such as the co-occurrence matrix and the run-length matrix did not show significant correlation with pathological findings. We supposed that this result had been reflected by the random distribution of collagenous tissue in the SFTs.The signal intensities displayed on T1-WI were mainly isointense and some were mildly hypointense or mildly hyperintense. Previous studies reported homogeneous isointensity 9 or mild10SFTs are well-defined masses located in various areas of the retrobulbar space. Zhang et al. reported that SFTs are always located in the superior retrobulbar region . Seven o4The homogeneity of enhancement was independent of the level of CD31 staining. SFTs’ neoplastic cells are negative for CD31 13. Howevρ = 0.75, p = 0.02) between the level of CD31 staining and the amount of collagenous tissue. This result might reflect the relation between neovascularization and growth of stroma.Kurtosis in T2WI and the level of CD31 staining showed a substantial correlation. The reason of this result was the strong correlation (The present study has some limitations. First, the number of subjects included in the study was small. Orbital SFTs are relatively rare and previous studies also analyzed a relatively small number of subjects. Therefore, we believe that large sample sizes are necessary. Secondly, the contrast-enhanced method varied in our cases. Dynamic studies play an important role in the differential diagnoses, as SFTs usually have a hypervascular nature.In conclusion, orbital SFTs showed a variable intensity on T2WI. Kurtosis in the histogram analysis on T2WI had a strong correlation with the amount of collagenous tissue.


Objective: To determine the diagnostic accuracy of fully automated electro-chemiluminescence immunoassay (ECLIA) anti-SARS-CoV-2 serological test for detection of past SARS-CoV-2 infection and to be used in seroprevalence surveys.Method: A total of 426 patients who had tested for anti-SARS-CoV-2 from August 1 to 31, 2020 were selected for the study. Informed consent was obtained and a questionnaire including the patient’s age, gender, symptoms, and past polymerase chain reaction (PCR) status was filled by the patient. Samples were analyzed for anti-SARS-CoV-2 antibodies on Roche Cobas e601.Results: The mean age of the patients was 42.43±16.67 years. One hundred and five (24.6%) were PCR positive, while 321 (75.4%) were PCR negative. Most patients were males 241 (56.6%) while 185(43.3%) were females. Over 185(43.3%) patients presented with symptoms, and the rest of the patients 241 (56.6%) were asymptomatic. Anti-SARS-CoV-2 had sensitivity 89.5%, specificity 99.06%, positive predictive value (PPV) 96.90%, negative predictive value (NPV) 96.6%, and positive likelihood ratio 4.26, while negative likelihood ratio 0.1. Diagnostic accuracy of anti-SARS-CoV-2 was 96.7% based on receiver-operating characteristic (ROC) curve analysis.Conclusion: Anti-SARS-CoV-2 is very useful for the detection of past COVID-19 infection; it can be proved helpful in the identification of post-COVID complications and actual disease burden in a population. Soon the world was grappling with a pandemic of massive proportions. Until now this disease has more than 27 million confirmed cases and about 890 thousand people have lost their lives in this pandemic [Towards the end of 2019, the Chinese city of Wuhan reported a cluster of pneumonia patients . Soon thpandemic .Whenever the scientific community has come face to face with a new infection knowledge regarding the source of the pathogen, mode of transmission, life cycle, pathogenicity, diagnostic tools, and treatment protocols are gradually perfected over a period of years. Because of the unprecedented rapid spread of COVID-19, the healthcare systems around the world were presented with many clinical and diagnostic challenges. Several newly developed screening and diagnostic test techniques started to fulfill the insatiable demand for rapid diagnosis across the globe. Real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) which detects SARS‐CoV‐2 in the throat and nasal swabs became the golden standard for diagnosis of COVID-19 . The perRapid serological test kits identifying different antibodies  generated a lot of interest because of their faster turn-around time, simplicity, and cost. However, the clinical worth of these tests depends mainly upon the performance characteristics like sensitivity, specificity, positive predictive value, and negative predictive value. The lack of adequate vigilance of serologic tests is concerning given that the commercially available serologic assays are highly variable, having different principles, detect variable antibody class, and have inconsistent sensitivities. Many rapid testing kits were based on lateral-flow immunoassay (LFIA) or enzyme-linked immunosorbent assay (ELISA) technique which have questionable results and have been called back from many centers . An addeRecently, fully automated electro-chemiluminescence immunoassay (ECLIA) has been introduced claiming to be highly sensitive and specific. The aim of our study is to determine the diagnostic accuracy of anti-SARS-CoV-2 (ECLIA technique) for diagnosis of past infection with COVID-19 using RT-PCR as the golden standard.This cross-sectional study was conducted from August 1 to 31, 2020 at the Clinical Chemistry section of National Medical Center Laboratory Karachi. A total of 426 consenting patients who visited the outpatient or emergency department of the hospital were included in this study. The study purpose was thoroughly explained to the patients. For all the enrolled patients, a questionnaire regarding age, gender, symptoms , and RT-PCR result for COVID-19 (report presented by the patient) in past 15-21 days were filled by the patient.g for 15 minutes to separate the serum. All antibody tests were performed using a fully automated Roche Cobas e601 immunoassay analyzer based on the ECLIA technique. This anti‑SARS‑CoV-2 assay uses a recombinant protein representing the nucleocapsid (N) antigen for the determination of antibodies  against SARS‑CoV‑2. The calibration and internal quality control were done according to the manufacturer’s recommendations.Venous blood samples were collected using a serum separator tube (SST). Tubes were centrifuged at 4000Patient samples were labeled as reactive (cut off >1.000) and non-reactive (cut off <1.000) as recommended by the manufacturer. Data were organized and entered on SPSS version 22 . Age was presented as mean ± standard deviation. The frequency of symptoms, gender, and history of positive or negative RT-PCR was calculated with Pearson's co-efficient p-values <0.05 taken as statistically significant.Diagnostic accuracy was presented in terms of sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios. ROC curve analysis was done to validate the performance of the anti-SARS-CoV-2 test.The average age of the patients was 42.43±16.67 years. The largest proportion of patients belonged to the 21-40 years age group 42.7% (n=182). Males constituted 56.6% (n=241) of the study group. The majority of patients were asymptomatic and tested negative for COVID-19 RT-PCR.Fever was noted to be the commonest symptom followed by body aches and sore throat as shown in Table Stratification analysis was performed and it was observed that the frequency of anti-SARS-CoV-2 reactivity was significantly high in people who were symptomatic or had COVID-19 RT-PCR positive in the past 14-21 days . However, no statistically significant difference was found in regard with age groups or gender , as represented in Table Anti-SARS-Cov-2 was found reactive in 22.8% (n=97) patients while 72.2% n=329) were found non-reactive. Sensitivity, specificity, PPV, NPV, positive likelihood ratio, negative likelihood ratio, and diagnostic accuracy are excellent as shown in Table 29 were fThe area under the ROC curve (AUC) for anti-SARS-Cov-2 was 0.943 which is considered an excellent value, as depicted in Figure Serum antibody tests are not for the detection of the presence of virus instead they tell us about the immunoglobulins (Ig) formed by B lymphocytes against the virus in the human body as a protective mechanism . With thThe total number of patients with positive PCR results was 105, out of which 97 (92.4%) patients presented with symptoms, the rest of the patients 07 (7.6%) were asymptomatic. These results are similar to another study from Karachi which showed only 9.0% were asymptomatic . A signiIn this study, anti-SARS-CoV-2 misidentified only 0.028% as false positives, while 99.06% were correctly classified as true negative, i.e., these patients also had negative PCR results, making this test highly specific. It is important to have very high specificity in order to minimize the risk of cross-reactivity with other viruses that cause common cold or influenza. The sensitivity of this test was found to be 89.5%. This slight low sensitivity may be attributed to variation in immune response and timings of sample collection. These observations differ from a study conducted by Muench et al. which showed better sensitivity and specificity of 99.5% and 99.08%, respectively. The stark gap in the noted sensitivity of this test calls for further validations .The high negative predictive value in this study provides a strong reassurance that a person has not suffered from COVID-19 in the near past. The sensitivity, specificity, PPV, NPV values, and accuracy of anti-SARS-CoV-2 were consistent with a study done by Lau et al. . AUC is Given the excellent performance characteristics of anti-SARS-CoV-2, it can be confidently used as a tool to assess community exposure and can help in making future health policies.However, the results of the current study can be improved upon by multicentre data collection and by longer duration for studying the performance of this test. In addition to this, the anti-SARS-CoV-2 test used in this study is approved only for qualitative analysis of antibodies which is superseded by quantitative assays helping in improved reporting in COVID 19 patients.Our study points towards high sensitivity, specificity, PPV, NPV, and excellent diagnostic accuracy of fully automated ECLIA anti-SARS-CoV-2 method, making it an ideal choice for identification of past COVID 19 infections and can be used as an efficient tool for mass screening and seroprevalence studies been done for detection of actual COVID-19 burden in a population.In conclusion, we can say that such a serological assay to detect antibodies against SARS-CoV2 can be used as an effective tool in our current response to the COVID-19 pandemic. As time progresses, we will learn more about our immune response to this virus, duration, and level of protective immunity provided by the development of antibodies against SARS-CoV-2. With existing knowledge about the advantages and limitations of this serological test, it can be safely recommended for use in public health studies and academic proposes, but additional data are required to build up on this foundation.


The sensitivity of reverse transcriptase polymerase chain reaction (RT-PCR) has been questioned due to negative results in some patients who were strongly suspected of having coronavirus disease 2019 (COVID-19). The aim of our study was to analyze the prognosis of infected patients with initial negative RT-PCR in the emergency department (ED) during the COVID-19 outbreak. This study included two cohorts of adult inpatients admitted into the ED. All patients who were suspected to be infected with SARS-CoV-2 and who underwent a typical chest CT imaging were included. Thus, we studied two distinct cohorts: patients with positive RT-PCR (PCR+) and those with negative initial RT-PCR (PCR–). The data were analyzed using Bayesian methods. We included 66 patients in the PCR– group and 198 in the PCR+ group. The baseline characteristics did not differ except in terms of a proportion of lower chronic respiratory disease in the PCR– group. We noted a less severe clinical presentation in the PCR– group . Hospital mortality (9.1% vs. 9.6%) did not differ between the two groups. Despite an initially less serious clinical presentation, the mortality of patients infected by SARS-CoV-2 with a negative RT-PCR did not differ from those with positive RT-PCR. The 2019 novel coronavirus causing Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) was reported for the first time in December 2019 in Wuhan, China . HoweverThe management of patients with typical clinical signs of SARS-CoV-2 pneumonia and typical chest CT imaging without RT-PCR results or with negative RT-PCR raises some pragmatic questions about, for example, isolation, orientation and prognosis . ClinicaWe used the prospective cohort called COVIDHUS, which includes all suspect patients of COVID-19 admitted to a university hospital during the COVID-19 epidemic. Our region was one of the most impacted by the pandemic in Europe, with nearly 3400 deaths and more than 10,000 patients infected at the end of May. This study included two cohorts of adult inpatients (>18 years old) admitted to the emergency department (ED) of a university hospital. We screened all the patients suspected of being infected with SARS-CoV-2 between 14 March and 16 April 2020 and selected those with a typical chest CT imaging. An RT-PCR test on nasal and pharyngeal swabs was performed in the emergency department, allowing us to identify two distinct patient cohorts: those with positive initial RT-PCR (PCR+ group) and those with negative initial RT-PCR (PCR− group). We selected patients in the PCR+ group by matching the patients by gender and age. The PCR−/PCR+ group ratio was one to three. In our study, COVID-19 was defined by the presence of the following three criteria: (a) epidemiological history ; (b) clinical manifestations ; (c) chest CT with typical lesions  . AccordiIn the ED, a chest CT scan and a nasopharyngeal swab were performed immediately at arrival in all patients admitted for COVID-19 clinical suspicion. The chest CT performed in the ED was interpreted by an experienced radiologist and classified as typical, indeterminate or incompatible with COVID-19. According to the European Society of Radiology and the European Society of Thoracic Imaging, the severity of lung damage was classified as minimal (<10%), moderate (10–24%), extensive (25–49%) and severe (≥50%) . PatientThe nasopharyngeal swab was collected by a trained team (ED head physician) according to a detailed video in a previously published study . QuantitData were collected from electronic medical records using a standardized consent report form. These data included epidemiological, clinical, laboratory and radiological data. The date of the onset of the symptoms  before the visit to the ED was collected. Clinical parameters were also recorded during the visit to the ED. Minimum oxygen saturation, blood pressure, heart rate, respiratory rate, oxygen requirement and Glasgow coma scale were recorded. The main medical histories that may affect prognosis were collected: pre-existing respiratory, cardiovascular or kidney diseases, active cancer, diabetes, hypertension and obesity ,20,21,22p-values and that the computed probabilities should not be confused with p-values. Probabilities near 1 or 0 suggest an effect, respectively, of a positive or negative difference. All computations were done with R 4.0 and JAGS software(s) with all the required additional packages.The data were analyzed using Bayesian methods. The data were described as frequency (%) for categorical data and with mean and standard deviation (SD) for quantitative data. To compare the two groups, we used mean comparison with Gaussian prior distribution for quantitative data and proportion comparison with Beta prior distribution for categorical data. Priors were defined before the study and based on mild assumptions derived from expert knowledge and available literature. Results are provided as point estimates of means or proportions difference with their associated 95% credibility interval. We computed the probability that the between-group difference is larger than 0 (Pr(diff > 0)). We remind the reader that Bayesian methods do not use Between 14 March and 16 April 2020, a total of 2329 patients visited our ED, of whom 1671 were suspected of having COVID-19. Among them, 680 patients had chest CT imaging typical of COVID-19. Of these, 66 had an initial negative RT-PCR and 447 a positive RT-PCR. We included all 66 patients with negative RT-PCR (PCR− group) and randomly matched them with 198 patients with initial positive RT-PCR (PCR+ group). In the PCR– group, 32 patients became positive during the length of their hospital stay (LOS) and 34 patients remained negative during their whole stay . The mea2 vs. 27.5 Kg/m2 (Pr(diff > 0 = 0.09). The most common comorbidities were hypertension  = 0.53), followed by diabetes  = 0.39) and active cancer or hemopathy  = 0.81)) without any significant difference. The number of patients with chronic respiratory disease was higher in the case group (PCR–) than in the PCR+ group  > 0.99). The baseline characteristics are presented in The mean respiratory rate and the mean oxygen need were lower in the PCR− group (23.8 breaths per minute (SD 5.7) vs. 27.1 breaths per minute (SD 9.1), Pr(Diff > 0) < 0.01 and 3.0 L/min vs. 4.4 L/min, Pr(diff> 0) < 0.01 respectively). Conversely, the proportion of patients with heart rate ≥120 bpm was higher in the PCR− group than in the PCR+ group  = 0.93). The biological and imaging characteristics are presented in Nevertheless, no difference was found in the in-hospital mortality rate, with 9.1% ) in the PCR– group vs. 9.6% ) in the PCR+ group (Pr(Diff > 0) = 0.50). Similarly, the LOS was not significantly different between the two groups (8.7 days (SD 7.4) in the PCR– group, vs. 9.8 days (SD 8.3) in the PCR+ group, (Pr(diff > 0) = 0.17). On the other hand, we noted that the proportion of patients requiring ICU admission tended to be lower in the PCR– group  = 0.11) .With more than 70% of patients consulting for suspected SARS-CoV-2 pneumonia during the COVID-19 epidemy, our hospital was hardly impacted. The study population was mostly a symptomatic hospitalized population. This study answers a pragmatic question in this specific population: Do patients with SARS-CoV-2 pneumonia and an initial negative RT-PCR test have a different outcome?During the epidemic phase, the diagnosis of COVID-19 was made by chest CT scan with a higher sensitivity than RT-PCR. In a recent meta-analysis, Kim et al.  showed tThere can be many reasons for a negative RT-PCR  ,8,9. In The mean age was 60 years, with a higher proportion of males (64%), as shown in previous studies ,30. The During visits to the ED, clinical respiratory parameters were significantly worse in the control group, with a mean respiratory rate of 27.1 vs. 23.8 breaths per minute. Patients in the control group required more oxygen than patients in the PCR– group. For other clinical parameters, no difference was observed. Li et al. recently suggested that patients with negative RT-PCR had the same clinical characteristics as patients with positive RT-PCR . Their sA limitation of our study could be a selection bias in defining COVID-19 with negative RT-PCR and typical chest CT lesions. Although we were in the middle of the pandemic, typical chest CT lesions could be another type of infection, even if it was a low probability. Half of the PCR– group turned positive after several nasal and pharyngeal swabs. The other half were considered as typical COVID-19 for physicians in this context of the high prevalence of the disease. This definition of COVID-19 diagnosis could not be used in case of lower prevalence and must be kept in mind for clinicians.Although single-centered and on a limited sample, our study answers a pragmatic question that arises in all emergency departments in the epidemic phase. Despite an initially less severe clinical presentation, less need for mechanical ventilation and ICU admission, mortality and length of stay of SARS CoV-2-infected patients with negative RT-PCR did not differ from patients with positive RT-PCR. The management of such patients should be the same in the ED as for those with PCR+.


To compare the plasma N-terminal pro-C-type natriuretic peptide concentrations of normotensive pregnant women, patients with mild preeclampsia, and patients with severe preeclampsia. U tests.  We collected venous blood samples from 25 normotensive pregnant women, 15 patients with mild preeclampsia, and 15 patients with severe preeclampsia. The women were at 30th to 40th weeks of gestation and in an age range of 20 to 35. The N-terminal pro-C-type natriuretic peptide levels were measured by ELISA. Statistical comparisons were made by one-way analysis of variance, Kruskal–Wallis, and Mann–Whitney P < 0.001), whereas there was no significant difference between those with mild preeclampsia and the normotensive groups (P > 0.05).  The median (interquartile range-IQR) values of the N-terminal pro-C-type natriuretic peptide were 6.48 (3.33) pmol/L in the normotensive women group, 7.37 (3.43) pmol/L in patients with mild preeclampsia, and 11.52 (6.13) pmol/L in patients with severe preeclampsia. The N-terminal pro-C-type natriuretic peptide was significantly elevated in the severe preeclampsia study group ( Our data indicate that the plasma concentration of the N-terminal pro-C-type natriuretic peptide is significantly increased in patients with severe preeclampsia, but not in patients with mild preeclampsia. The severity of preeclampsia may be related to the circulating levels of the N-terminal pro-C-type natriuretic peptide concentrations. The natriuretic peptide family is composed of three principal peptides sharing the same 17 amino acid ring structure: A-type  natriuretic peptide, B-type (brain) natriuretic peptide, and C-type natriuretic peptide . The A-tThe C-type natriuretic peptide was the third member of this family to be identified. The C-type natriuretic peptide is a locally acting peptide in an autocrine/paracrine fashion. It is present at very low concentrations in the plasma, as a consequence of its rapid clearance from the circulation . It has Preeclampsia and eclampsia are two of the hypertensive disorders of pregnancy. The pathophysiological mechanisms in the development of these disorders are not clear yet although it is well known that endothelial dysfunction has a fundamental role . As far   Systolic blood pressure of 160 mm Hg or higher or diastolic blood pressure of 110 mm Hg or higher on two occasions at least 4 hours apart while the patient is on bed rest (unless antihypertensive therapy is initiated before this time)  Thrombocytopenia   Impaired liver function as indicated by abnormally elevated blood concentrations of liver enzymes , severe persistent right upper quadrant or epigastric pain unresponsive to medication and not accounted for by alternative diagnoses, or both  Progressive renal insufficiency   Pulmonary edema  New-onset cerebral or visual disturbancesA total of 55 pregnant women followed in the Department of Obstetrics and Gynecology, Zeynep Kâmil Women and Children Diseases Education and Research Hospital, Turkey, were enrolled in this study. 15 patients with severe preeclampsia and 15 patients with mild preeclampsia were selected for the preeclamptic group. A written informed consent was obtained from all women. All women selected were between the ages of 20 and 35 and had singleton pregnancies. Those with diabetes, cardiac or renal disease, and chronic hypertension were excluded from the study. Also, women diagnosed with HELLP syndrome were excluded from the study. All women were also in their third trimester of gestation. None of the women was in labor. Arterial blood pressures were recorded for at least two different times 4 hours apart when the patient is on bed rest from the right arm using a standard sphygmomanometer with an appropriate cuff size by the same clinician. Korotkoff sound K5  was used to determine diastolic blood pressure. Preeclampsia is defined by the minimum criteria of blood pressure ≥140/90 mm Hg after 20 weeks of gestation and proteinuria 300 mg/24 hours or 1+ dipstick, according to the report by the Report of the American College of Obstetricians and Gynecologists' Task Force on Hypertension in Pregnancy . Twenty 2EDTA). The blood samples of patients with preeclampsia were collected before they received any medication. Immediately after sampling, plasma samples were separated by centrifugation at 2000 × g for 15 min at 4°C and frozen at −80°C until assayed. The N-terminal pro-C-type natriuretic peptide levels in these plasma samples were measured with the ELISA method using the N-terminal pro-C-type natriuretic peptide EIA Kit . The lower detection limit was 0.55 pmol/L. The intraassay variations were 5-6% for a concentration range of 5.1–20 pmol/L, and the interassay variations were 1% for a concentration range of 5–20 pmol/L as stated by the manufacturer. All of the samples were run in a duplicate manner in the same assay. The laboratory staff were blinded to the clinical condition of the women in the study groups.One venous blood sample (10 mL) was collected from the patients with preeclampsia and the healthy controls into tubes containing ethylenediaminetetraacetic acid . Repeated measures of one-way analysis of variance (ANOVA), Kruskal–Wallis, and Mann–Whitney Our study protocol was approved by the institutional review board.P < 0.05) (As shown in  < 0.05) . The disP < 0.001) and mild preeclampsia (P < 0.01) study groups, whereas there was no significant difference among the mild preeclampsia and normotensive study groups (P > 0.05). The median (IQR) values of the N-terminal pro-C-type natriuretic peptide were 6.48 (3.33) in the normotensive women group, 7.37 (3.43) in patients with mild preeclampsia, and 11.52 (6.13) in patients with severe preeclampsia. The spread of data among the study groups and the control group is displayed in With regard to the biochemical findings presented in Nppc) is associated with hypertension in a Japanese study [The C-type natriuretic peptide was the third member of the natriuretic peptide family to be discovered. The natriuretic peptide family has important effects on the control of blood pressure, volume, and the management of the renal function . The C-tse study . Allelicse study . Furtherse study . The C-tse study  and adrese study , aldostese study , and vasse study . Preeclase study . The secse study .In our study, we compared the plasma N-terminal pro-C-type natriuretic peptide concentrations of 55 subjects, composed of 25 normotensive healthy pregnant women and 30 pregnant women with preeclampsia, all of which were in their 30–40 weeks of gestational ages. The preeclamptic study group was further divided into severe and mild preeclamptic groups, each composed of 15 patients, discriminated according to the criteria defined in the report by the Report of the American College of Obstetricians and Gynecologists' Task Force on Hypertension in Pregnancy . We founOur data indicate that the severity of preeclampsia could be related to the circulating levels of the N-terminal pro-C-type natriuretic peptide. Espiner et al. indicated that preeclampsia and GHT with SGA were associated with raised maternal plasma NTproCNP . Since tSince we know that C-type natriuretic peptide infusion drops blood pressure, it may also find a therapeutic use in treatment of hypertension in pregnant women. It is evident that further studies are needed to clarify the prognostic and therapeutic role of the C-type natriuretic peptide in preeclamptic patient population.


Effective nose-to-brain delivery needs to be developed to treat neurodegenerative diseases. Regulating miR-124 can effectively improve the symptoms of ischemic brain injury and provide a certain protective effect from brain damage after cerebral ischemia. We used rat models of middle cerebral artery occlusion (t-MCAO) with ischemic brain injury, and we delivered RVG29-NPs-miR124 intranasally to treat neurological damage after cerebral ischemia. Rhoa and neurological scores in rats treated by intranasal administration of RVG29-PEG-PLGA/miRNA-124 were significantly lower than those in PEG-PLGA/miRNA-124 nasal administration and RVG29-PLGA/miRNA-124 nasal administration group treated rats. These results indicate that the nose-to-brain delivery of PLGA/miRNA-124 conjugated with PEG and RVG29 alleviated the symptoms of cerebral ischemia-reperfusion injury. Thus, nasal delivery of RVG29-PEG-PLGA/miRNA-124 could be a new method for treating neurodegenerative diseases. The focus of cerebral apoplexy treatment is to protect normal brain tissue and promote the recovery of the ischemic penumbra near the infarcted area  in nerve cells -poly(lactic-co-glycolic acid)), MePEG-PLGA, and PLGA were purchased from Jinan Daigang Biomaterial Co., Ltd. Spermidine and DiR  were purchased from Sigma-Aldrich . RVG29-Cyspeptide and PLGA-maleimide (MAL) were purchased from Changchun Xinjinji Biological Technology Co., Ltd. The miR-124 mimic was purchased from Shanghai GenePharma Co., Ltd.2.1.2.g for 45 min and washed 3 times with deionized water. The maleimide-thiol interaction was used to couple the RGV29 peptide to the surface of the NPs. The excess RGV29 peptide was incubated for 8 h, and the unreacted peptide was removed by centrifugation . The same steps were used to prepare RVG29-modified PLGA nanoparticles and blank nanoparticles without miR-124. DiR-labeled nanoparticles were generated by adding DiR to the oil phase, and the preparation process was the same as described above. The particle size and zeta potential of the nanoparticles were measured by dynamic light scattering (DLS) using a Zetasizer 3000 . Nanoparticles were imaged using a transmission electron microscope . The encapsulation efficiency of miRNA loading was tested with a Quant-iT™ RiboGreen kit.PEG-PLGA nanoparticles loaded with miR-124 were prepared by a double emulsion method. MiR-124 was mixed with spermidine in a 10:1 N/P ratio in RNase-free water to form a complex. Eighteen milligrams of PEG-PLGA and 2 mg of maleimide-PEG-PLGA were separately dissolved in 2 mL of dichloromethane (DCM), and then, the spermidine/miR124 complex was added to the above DCM solution and sonicated for 60 s to form a primary emulsion. The primary emulsion was added to 20 mL of a 2.5% PVA (w/v) aqueous solution and stirred, and the organic solvent was removed by rotary evaporation. The nanoparticles were concentrated by centrifugation at 21,000 × 2.1.3.Rats with MCAO were intranasally instilled with PEG-PLGA NPs and RVG29-PEG-PLGA NPs labeled with the dye 1,1′-dioctadecyl-3,3,3′3′-tetramethylindotricarbo-iodine cyanine (DiR). Two hours later, the heart was perfused with normal saline, and the brain and internal organs were collected. Biofluorescence imaging systems  were used to capture and analyze tissue fluorescence images of the brain and related organs.2.2.2.2.1.Adult SD rats  were selected and provided by Liaoning Changsheng Biotechnology Co., Ltd.  2015-0001). The controlled temperature was 23 ± 2 °C, the relative humidity was 54 + 2%, and water was freely available. All operation procedures were in accordance with the requirement for ethical approval by the Ethics Committee of the School of Pharmaceuticals Sciences, Jilin University. All animal studies were strictly conducted under the guidelines of the ‘National Animal Management Regulations of China’ and were approved by the Animal Ethics Committee of the School of Pharmaceuticals Sciences, Jilin University.2.2.2.Supplementary materials).We used the modified line embolus method for 2 h to generate the rat model of focal cerebral ischemia model (MCAO). Briefly, the rats were administered 10% chloral hydrate, and the common carotid artery, internal carotid artery, and external carotid artery were exposed. A surgical 4-0 nylon line was used to ligate the artery from the external carotid artery to the internal carotid artery (18.5–19.5), and the right middle cerebral artery was obstructed, inducing MCAO for 2 h. Then, the nylon line was removed. The neurological function scores were determined at 24 h, and the animals were divided into groups , and miR-124 nasal administration groups, including the PEG-PLGA/miRNA-124 nasal administration group, the RVG29-PEG-PLGA/miRNA-124 nasal administration group, and the RVG29-PLGA/miRNA-124 nasal administration group. Six animals were in each group. In each group, the administration was started 7 days before the modeling, and on the 7th, the 5th, the 3rd, and the 1st day of the modeling, each rat was nasally administered two drops of miRNA-124 at a concentration of 5 µg/mL aqueous solution. All rats were anesthetized and sacrificed after 3 days. All procedures complied with the relevant animal ethical regulations.2.2.4.0 score: no symptoms of nerve injury;1 points: cannot fully extend the contralateral front claw;2 points: rear-end turn to the opposite side;3 points: slouching to the opposite side;4 points: unable to walk spontaneously, loss of consciousness.After 24 h of ischemia/reperfusion in the rats, the Zea Longa scoring method was used to score the MCAO reperfusion model, and the scoring criteria were as follows:2.2.5.unable to stay on the beam;1, able only to stay on the beam;2, tried to traverse the beam but fell:3, traversed the beam with more than 50% hindlimb foot slips;4, traversed the beam with fewer than 50% foot slips;5, traversed the beam with only one slip;6, traversed the beam with no slips.Fine motor coordination and balance were assessed by the beam walking assay. This test is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the rotarod test. A beam-walking test was applied to assess motor coordination and balance control of the forelimbs and hindlimbs to evaluate neurological functional recovery in the rats after ischemia. Beam walking tests were used to assess fine motor coordination 24 h after MCAO. Briefly, each rat was trained to traverse a horizontal beam 2.5 cm wide, 80 cm long and 60 cm above the floor. Rats were motivated to cross the beam by placing their home cage at the other end. The behavioral response was scored as follows:2.2.6.Cerebral infarction volume in rats was measured by TTC staining. The rats were anesthetized, and the cerebellum and olfactory bulb were removed. After the samples were frozen at −20 °C for 30 min, continuous coronal sections of the brain tissue were obtained at 2 mm intervals, stained with 1% TTC solution at a constant temperature of 37 °C for 30 min, and stained with regular rotation. The infarcted areas of the brain were white, and the noninfarcted areas were red. The section was photographed and analyzed by ImageJ software. Cerebral infarction area (%) = cerebral infarction area/whole brain area ×100%.2.2.7.Some of the rats in each group were subjected to heart perfusion with paraformaldehyde. After the brain tissue was fixed, the brain was stored in a 4% paraformaldehyde solution for subsequent immunofluorescence detection. After the brains of the remaining groups of rats were collected, immunofluorescence was used to assess RhoA and GAP43. A portion of the brain tissue was directly frozen in liquid nitrogen for PCR and Western blot analyses of RhoA and GAP43, and RhoA and GAP43 were analyzed.2.3.One-way analysis of variance and Newman–Keuls tests were used for the statistical analyses. Statistical analysis was performed using GraphPad Prism 6.0 software. Quantitative analysis of the WB bands was performed using ImageJ analysis software.3.3.1.Transmission electron microscopy (TEM) analysis showed that the RVG29-PEG-PLGA/miRNA-124 nanoparticles appeared regular and spherical . The ave3.2.In order to assess the aggregation of nanomaterial coated miRNA-124 in the brain of ischemic stroke, after successful modeling of ischemic stroke, three kinds of nanoparticles  were administered to the nasal mucosa, and the fluorescence intensity in the brain was detected 4 h after administration. The results showed that rvg29-peg-plga/miRNA-124 had the highest accumulation of fluorescence intensity in the brain compared with the other two groups. The accumulation of peg-plga/miRNA-124 and rvg29-plga/miRNA-124 in the brain was small and the differences were not significant. It indicated that the selection of nanoparticle ascomaterials and the existence of modification groups can help the nanoparticle to reach the target position better. The experimental results showed that peg-plga modified with RVG29 could be used as a nanocarrier to enhance the aggregation of the drug in the brain .3.3.p < .05), showing that the miRNA-administered groups displayed improvements in brain function compared to the model group. The neurological deficit scores of RVG29-PEG-PLGA/miRNA-124 nasal administration group were significantly lower than those of the ischemic brain model group (p < .05) but higher than those of the sham operation group. The nanoparticle group-administered miRNA by nasal inhalation had the lowest neurological defect score, indicating the excellent therapeutic effect of this treatment . Compared with that in the control group, the percentage error on the beam balance was significantly reduced the PEG-PLGA/miRNA-124 nasal administration group  Compared with that in the PEG-PLGA/miRNA-124 nasal administration group, the percentage error on the beam balance was significantly reduced RVG29-PEG-PLGA/miRNA-124 nasal administration group . These results showed that the nasal inhalation method is very effective and fast for treating brain diseases and can significantly reduce dyskinesia after cerebral infarction. The carrier form of the nanoparticles and miRNA had the best pharmacodynamic effect , the percentage error on the beam balance in the ischemic brain injury group was significantly higher (1.67 ± 0.82, c effect .3.5.TTC tests were used to verify the effect of nasal administration of RVG29-NPs-miR124 on cerebral ischemia-reperfusion. TCC staining was used to assess the size of the cerebral infarction area; the lesion area of the cerebral infarction was not stained white. The MCAO treatment lasted 2 h, and the brain infarct size and the neurological score were measured at 24 h after reperfusion. As shown in Figure, the infarct area of the model group and the RVG29-NP group was basically the same, and the neurobehavioral score did not show significant improvements, indicating that the nanoparticles themselves had no therapeutic effect; however, the administration of RVG29-NP/miR-124 improved the neurobehavioral score and significantly reduced the infarct size. Naked miR-124 had an effect on the infarct area, but this effect was not optimal. These results suggest that nanoparticles can protect miR-124 from nasal cilia or related enzymes to a certain extent .3.6.RhoA is the main scaffold protein in excitatory synapses and postsynaptic densities and has been characterized as one of the most abundant scaffold proteins in excitatory neurons. GAP43 is a marker of neurons. Quantitative analysis showed that the fluorescence intensities of RhoA and GAP43 in the RVG29-NPs-miR124 group were significantly higher than those of the MCAO model group. primarily due to the high-affinity interaction of the RVG29 conjugated to the nanoparticle surface with the neuron surface receptors, and the nanoparticle protected miR-124 from being degraded and could reach the target site through nasal inhalation .3.7.Changes in protein expression were detected by Western blots. The results showed that the expression levels of RhoA and GAP43 increased in the MCAO group. The RVG29-NPs-miR124 group exhibited significantly decreased expression of RhoA in the Area of cerebral infarction. Furthermore, GAP43 expression was increased compared with that in the other groups. In the PEG-PLGA group and the RVG29-PLGA group, the upregulation of GAP43 was weaker than that in the RVG29-PEG-PLGA group. These results showed that the modification of RVG29 and PEG is important to enhance the nasal inhalation of the PLGA nanoparticles .4.Ischemic cerebral apoplexy is the second most fatal disease worldwide after cardiovascular disease Heuser, . This coWhen an ischemic brain injury occurs, the cytoskeleton needs to be regulated after the nervous system is damaged to maintain the dynamic stability between different components of neurons to achieve the integrity of the structure and function of nerve cells (Fujita & Yamashita, After cerebral ischemia, RhoA expression increases in the brain, strengthening the polymerization of actin in the growth cone and driving axial stretch to cause synaptic contraction of dendritic and spinous processes in nerve cells (Mulherkar et al., Caenorhabditis elegans (Lee et al., With the development of research, interventions for various diseases at the genetic level are increasingly recognized. MiRNAs were originally discovered in in vivo, with potential regulatory effects on postmitotic neuronal activity, such as synaptic plasticity and memory formation (Maiorano & Mallamaci, Our results showed that nasal administration of miRNA-124 for ischemic brain injury had significant effects. As an abundant neuron-specific miRNA in the brain, under different conditions, miR-124 has been identified as a promoter of neuronal generation in vitro and RhoA has a negative regulatory effect on neuronal synapse development (Walchli et al., GAP43 is a marker of neurite elongation and synapse formation (Rosskothen-Kuhl & Illing, The nasal-to-brain drug delivery pathway occurs via nasal breathing and the olfactory area through the intracellular exocytosis of cells or neurons or the intercellular pathway along the cells near the olfactory nerve, and the transcellular pathway across basal epithelial cells mediates drug delivery to the brain (Hong et al., 5.In this study, we verified our hypothesis that miR-124 encapsulated in RVG29-PEG-PLGA nanoparticles and administered through the nose, which allowed the drug to enter the brain and affect the expression of RhoA, could produce neuroprotective effects. The modification of RVG29 and PEG improved the effect of the PLGA nanoparticles as the delivery carrier through nasal inhalation. Therefore, we believe that supplementation with RVG29 and nasal delivery of miR-124 can indeed contribute to the recovery of neurological function after cerebral ischemia.


The use of electronic cigarettes (e-cigarettes) in adults is increasing globally, and similar trends may be observed in the young population. Our objective was to estimate the awareness and use of e-cigarettes among the students from two comprehensive universities in Shanghai, China, and to identify the factors that may influence their decision to use e-cigarettes and their possible adverse effects.An online cross-sectional survey was conducted among the students of Shanghai Jiao Tong University and Fudan University. A total of 869 students , mean age 21.09 years (SD=2.44), were recruited in 2018. Multivariate binary logistic regression analyses were performed to explore the associations between ever e-cigarette use and influencing factors.Of the responding students, 88.4% were aware of e-cigarettes, 4.6% had used e-cigarettes at least once in their lifetime, and 1.7% were current e-cigarettes users. Males and smokers were more likely to use e-cigarettes (ever used e-cigarettes even once) than females  and non-smokers . University students were easily motivated to use e-cigarettes when their peers also used them, and the risk ratio was 4.15 (95% CI: 2.11–8.19) compared with if their peers never used e-cigarettes. The major factors found to motivate university students to use e-cigarettes were the belief that e-cigarettes were less harmful or not harmful (55.0%) and the perception that e-cigarettes were helpful to quit smoking (37.5%). The survey also indicated that 72.4% of the respondents heard about e-cigarettes from television advertisements, 42.7% from websites online, and 41.2% from their parents and friends.University students who were males, cigarette smokers and whose peers used e-cigarettes were more likely to use e-cigarettes. The use of traditional cigarettes should be controlled strictly in order to reduce the likelihood of e-cigarette use among university students. E-cigarettes consist of a power source, a heating element, and a tank or cartridge containing an ‘e-liquid’ in which there are nicotine (or no nicotine) and flavors dissolved in glycerol-propylene glycol2. Inhaling nicotine through e-cigarettes without combustion is believed to be safer than traditional tobacco burning by eliminating harmful combustion products, including tar and carbon monoxide3.Electronic cigarettes (e-cigarettes), also known as electronic nicotine delivery systems, are hand-sized battery-operated devices, which were originally designed to retain the look and feel of traditional cigarettes by delivering nicotine vapor to the user4. The International Tobacco Control (ITC) China survey showed that the percentage of smokers who had heard of e-cigarettes increased from 29.0% to 60.0%, and those who had tried e-cigarettes from 2.0% to 11.0%, in the period 2009 to 20145. According to the China Adult Tobacco Survey 2015, 52.3% of the population aged 15–24 years were aware of e-cigarettes and 4.1% had tried e-cigarettes, which were both higher than in other age groups6. In 2015, one mobile app-based survey in China, which recruited 2042 adolescents aged 12–18 years, showed that 89.5% of the surveyed adolescents were aware of e-cigarettes and 26.4% of the respondents had used e-cigarettes7. But we inferred the numbers might be higher than the true figures since people who were interested in e-cigarettes were more likely to participate in the online questionnaire. Another report indicated that 45.0% of middle school students had heard of e-cigarettes and 1.2% of participants had used e-cigarettes in the last 30 days8. A cross-sectional study including 5482 university students in Texas indicated that perceptions of harm and addictiveness of e-cigarettes were lower than those for conventional cigarettes among university students9. Furthermore, young adults are more likely to be aware of and use e-cigarettes if current trends in e-cigarette advertising continue10; common e-cigarette advertising is designed to imply safety and smoking cessation, and incorporates health claims not supported by medical evidence12.The device was first introduced to the Chinese domestic market in 2003 as an aid for smoking cessation and a replacement for regular cigarettes14 encouraged the increase in the adoption of e-cigarettes and the misconceptions of the benefits of using e-cigarettes, especially among young adults12, since e-cigarettes are marketed to appeal to youth15. However, few studies have examined awareness and use of e-cigarettes among Chinese university students and the predictors of e-cigarette use, which are crucial to understanding the use of e-cigarettes by college students and the associated motivational factors. Therefore, our objective was to estimate the awareness and use of e-cigarettes among university students in Shanghai and to explore the possible predictors that encourage e-cigarettes use.In China, the lack of regulations for e-cigarette use and the unrestricted practice17. We adjusted parts of the questionnaire according to the investigated subjects and our experience, including the baseline information and the cognition on the health hazards of e-cigarettes. The survey was made available in these two universities through an application called ‘So Jump’ which can design, distribute and recover questionnaires online, between March and April 2018. Taking a class as the unit, we used the cluster sampling to collect information about awareness and use of e-cigarettes. The survey was completely anonymous. In total, 1046 participants were selected, while 869 university students  completed the questionnaire with a response rate of 83.1%. Ethical approval was not necessary according to the Ethics Committee of School of Public Health, Shanghai Jiao Tong University School of Medicine, as this study was not an experimental study.A cross-sectional survey was developed among undergraduates and graduate students who studied in Shanghai Jiao Tong University and Fudan University, two top universities in Shanghai, in the summer semester of 2018. The survey questionnaire was designed based on the standard Chinese urban adult tobacco questionnaire and the network surveyWe gave the questionnaire, named ‘Survey on the Awareness and Use of E-cigarettes’, to the college counselors who distributed it to the entire class of students through the ‘So Jump’ application.The respondents were first asked about their age, gender, specialization and smoking experience (categorized as smokers and non-smokers). Participants were divided into three groups according to grade: junior students, senior students, and graduate students. Next, we assessed their awareness of e-cigarettes through the question: ‘Have you ever heard of e-cigarettes?’. Participants who answered ‘Yes’ were further asked: ‘Where have you heard of e-cigarettes?’ (multiple choice). All respondents were asked about their perceptions of e-cigarettes. The first question in this part was about student’s general perspective of e-cigarettes. The choices were: ‘Smoking cessation products’, ‘Environmentally friendly (producing less harmful substance to environment) alternatives to traditional cigarettes’, and ‘Electronic product’. The students then answered several questions about health hazards, addiction, and smoking cessation help of e-cigarettes. Students who thought e-cigarettes were environmentally friendly alternatives to traditional cigarettes, healthier and less addictive than traditional cigarettes were considered to have a positive opinion of e-cigarettes. Those who thought e-cigarettes could help quit smoking and avoid secondhand smoke, and contained no carcinogens, were also considered to have a positive opinion of e-cigarettes.Respondents who answered ‘Yes’ to the question ‘Do you vape e-cigarettes currently?’ were categorized as current e-cigarette users and those who answered ‘Yes’ to ‘Have you ever tried e-cigarettes (even once)?’ were categorized as ever e-cigarette users. Finally, we asked students whether there was anyone in their social circle that used e-cigarettes and whether they experienced any adverse effects from e-cigarettes.The final version of our questionnaire comprised 23 questions, 19 of which were single-choice questions and 7 questions that provided a ‘not sure’ option. The questionnaire is given in the Supplementary file.Data were analyzed using SPSS version 22.0 and were verified for normality of distribution and equality of variances. The quantitative variables were presented as mean and standard deviation (±SD) and qualitative data were described as frequency and percentage (%). Chi-squared tests were performed to compare awareness ratio of e-cigarettes and prevalence of ever use by demographics and other basic information. We then conducted unadjusted and multivariate logistic regression analyses to explore the risk factors of ever e-cigarette use, with an odds ratio calculation. In multivariate logistic regression analyses, ever e-cigarettes use was a dependent variable. Independent variables included demographics and variables identified by unadjusted logistic regression analysis that had a statistically significant association with ever e-cigarette use. Significant test was a bilateral test and the level of statistical significance was set at p<0.05 for all the analyses.In all, 869 participants out of 1046 (83.1%) completed all survey questions. Among the valid respondents, 412 (47.4%) were males and 457 (52.6%) were females, with an average age of 21.09±2.44 years. There were 235 (27.0%) junior students, 398 (45.8%) senior students, and 236 (27.2%) graduate students. As for their specialization, the majority of respondents (44.4%) were medical students, 24.3% were engineering students, 23.1% were science students, and 8.2% were liberal art students. In total, 56 (6.4%) respondents reported cigarette use, with median years of smoking 2 years (IQR:1–4 years).Respondents who answered ‘yes’ to the question ‘Have you ever heard of e-cigarettes?’ were considered to be aware of e-cigarettes. In our survey, the majority (88.4%) of participants were aware of e-cigarettes . Of the graduate students, 92.8% were aware of e-cigarettes, while 88.4% of the senior students and 83.8% of the junior students were aware of them, with a significant difference (p=0.01). Of the medical students, 92.7% were aware of e-cigarettes, which was higher than that of the engineering students (83.9%) and science students (84.6%) (p=0.003). Cigarette smokers (96.4%) were more aware of e-cigarettes than never smokers (87.5%) in our study (p=0.004) .The majority (72.4%) of students heard about e-cigarettes from television advertisements and about half of the students (42.7%) knew about e-cigarettes through the internet. Elsewhere, university students knew about e-cigarettes directly through their parents, friends and classmates (41.2%), or indirectly from other ways (27.7%) such as a micro-blog and Wechat , from stores and supermarkets (11.0%), pharmacies (2.5%) and other channels (2.4%) .The majority of participants had a positive opinion of e-cigarettes. Most respondents thought that e-cigarettes were healthier (78.0%) and less addictive (63.1%) than regular cigarettes. Most (72.6%) declared that e-cigarettes were used as an environmentally friendly alternative to traditional cigarettes and 81.0% thought that e-cigarettes were helpful for quitting smoking. More than half of the respondents reported that e-cigarettes could help avoid secondhand smoke and contained no carcinogens or chose ‘not sure’. Furthermore, they believed that e-cigarettes would replace traditional cigarettes partly or completely in the future (70.9%) .We also addressed how perception of e-cigarettes may be influenced and found that men and junior students were more likely to perceive e-cigarettes as being less harmful than cigarettes and more helpful in quitting smoking . Interestingly, smokers were more likely to perceive e-cigarettes as not helpful for quitting smoking compared with never smokers (p=0.009). Men (p<0.001) and senior students (p<0.001) were more inclined to believe that e-cigarettes could help avoid secondhand smoke. In addition, men, junior and senior students, and non-medical students, were more likely to perceive e-cigarettes as having no carcinogens  .Only 4.6% of participants reported that they had ever used e-cigarettes, 1.7% of participants were current e-cigarette users and two people (0.2%) had been using e-cigarettes every day. The ever e-cigarette users reported various reasons for their behavior such as ‘less harmful than traditional cigarettes’ (55.0%), ‘helpful for quitting smoking’ (37.5%), ‘diverse flavors’ (27.5%), ‘avoiding secondhand smoke’ (22.5%), ‘fashionable’ (17.5%), ‘can be used in smoke-free places’ (12.5%), and ‘alternatives to traditional cigarettes (10.0%)’.We also established the predictors of ever e-cigarette use by unadjusted and multivariate logistic regression analyses . UnadjusMultivariate analysis adjusted for age, gender, specialization, use of traditional cigarettes, use of e-cigarettes in social circle, and the items: can e-cigarettes help for quitting smoking, can e-cigarettes replace traditional cigarettes, do you think teenagers are more interested in e-cigarettes, and addiction of e-cigarettes compared with traditional cigarettes. It only indicated that cigarette smokers and the students whose peers used e-cigarettes were more likely to use e-cigarettes  .Among respondents who had used e-cigarettes, 33 respondents reported no adverse reactions, 4 respondents felt thirsty (10.0%), 3 respondents developed a throat irritation cough (7.5%), 2 respondents had nausea (5.0%), and 1 respondent had headache (2.5%).18. However, the figure was significantly higher than that of the 2015 China Adult Tobacco Survey (40.5%), which was conducted among adults aged ≥15 years6. Previous studies also reported that the awareness of e-cigarettes among high or junior high school students was 70.6% and 46.7%, respectively20. These results might be due to the following reasons. On the one hand, university students live in a comparatively free social environment. They are more likely to have access to television advertisements and the internet and are willing to try fashionable and novelty things such as e-cigarettes21, which was reported as the major way in which awareness of e-cigarettes was created in our study. On the other hand, young people were one of the main marketing target groups of the tobacco industry15. E-cigarette companies marketed their products to young people by promoting flavors and a modern stylish design. They also sponsored free samples and hired young attractive women to promote products at youth-oriented events22.Awareness of e-cigarettes was equal to those in United States universities (89.0% and 87.0% in 2017)24. Moreover, we also explored the perception of e-cigarettes among respondents and found that most thought that e-cigarettes were less harmful than regular cigarettes and e-cigarettes were an alternative to traditional cigarettes. More than four-fifths of participants believed that e-cigarettes could help quit smoking. Many studies found that e-cigarette users perceived e-cigarettes as being a healthier alternative to regular cigarettes, consistent with our study25. Although there were fewer toxic chemicals and of less concentration in e-cigarette aerosols than in traditional cigarettes, the unsubstantiated belief in the positive health effects of e-cigarettes might be harmful to the young population, especially the young never smokers26. As such, further research on the health effects of e-cigarettes is needed. Furthermore, a meta-analysis of 38 studies implied the odds of quitting traditional cigarettes were 28.0% lower in those who used e-cigarettes than in those who did not27. Another study indicated that e-cigarette ever use was associated with a higher risk of starting smoking and with a lower risk of quitting smoking among nursing students28. Loukas et al.29 conducted a study among university students and reported that cigarette users who also used e-cigarettes were no more likely than cigaretteonly users to attempt to quit smoking cigarettes. The World Health Organization (WHO) also revealed that there was insufficient evidence showing that e-cigarettes can help quit smoking, but, in fact, led to dual use of e-cigarettes and cigarettes30. So, we should be concerned about the gateway and renormalization effects. The former refers to two possible situations where e-cigarettes may aggravate initiation of nicotine use among non-smokers and then non-smokers who are addicted to nicotine, through e-cigarettes, may switch to cigarette smoking. The renormalization effect means the alternative flavors and fashionable features of e-cigarettes may attract more non-smokers to cigarettes30.We also discovered that the reasons of ever e-cigarette use in university students in China were different from those in the United States. Our study showed that most of respondents used e-cigarettes because they were perceived as less of a hazard, assisted in smoking cessation, and there was no secondhand smoke. The university students in the United States considered using e-cigarettes for their novelty rather than as a cessation aid18 were 50% and 26.7%, and the prevalence of current e-cigarette use were 10.0% and 6.4%, respectively. Also, the prevalences of ever use and current use of e-cigarettes among French students were 23.0% and 5.7%, respectively, higher than in our investigation31. However, in China, the rates of e-cigarette use have been rising. The survey conducted in Tianjin showed that 2.3% of participants had ever used e-cigarettes and 0.5% were current e-cigarette users32, consistent with the results of the 2015 China Adult Tobacco Survey6. Additionally, several studies conducted among adolescents suggested that higher rates of e-cigarette use than adults19, so we need to continue to monitor access to e-cigarettes among the young population.The prevalence of ever e-cigarette use and current e-cigarette use in our study were lower than those of studies in the US. The prevalence of ever e-cigarette use in two US studies33, which suggests that social interactions are an important way for sharing information about e-cigarettes and motivate more people to use e-cigarettes34. Hall et al.34 mentioned that 45.0% of participants wished to talk about e-cigarettes and nearly a third of participants had ever recommended e-cigarettes to others. Most of the participants recommended e-cigarettes for health reasons and smoking cessation34. We can see that social networks are a vital way information regarding e-cigarettes is spread and to make people more aware of and to try e-cigarettes34; we could also utilize such networks to do the opposite with accurate knowledge about the harms of e-cigarettes.We found that cigarette use and peer cigarette use would increase the chance of ever e-cigarette use. Smokers were more likely to connect primarily to other smokers and separate from non-smokers35, weight control behaviors36, which are likely to affect the chance of using e-cigarettes. Second, the subjects were recruited through the internet and so we did not avoid selection bias. Third, we explored the correlation between ever e-cigarette use and influencing factors rather than current e-cigarette use considering sample size. These categorizations may result in some misclassifications of e-cigarette user status. The sample size of our study was limited and should be enlarged in future studies.There are several limitations in our study. First, we did not consider lifestyle and social factors such as alcohol intake, energy drink consumption, and social frequencyOur survey of university students suggests that 88.4% had heard of e-cigarettes but the prevalence of ever e-cigarette use was relatively low. Of concern, the majority of university students heard about e-cigarettes from advertisements and websites, and most held the belief that e-cigarettes were safer and less addictive than traditional tobacco cigarettes. Prevention campaigns via social media thus appear to be an effective mechanism for influencing trends when targeting university students. Male gender, smoking experience and having peers who use e-cigarettes were associated with ever e-cigarette use.Although there are fewer toxic chemicals and of less concentration in e-cigarette aerosols than tobacco cigarettes, the long-term health effects of e-cigarettes are still unclear. Prospective surveys should be directed to addressing the potential harms and gateway effects to other tobacco products, from smoking e-cigarettes.The authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none was reported.This work was supported by the National Natural Science Foundation of China (Grant No. 81602929).Not commissioned; externally peer reviewed.Click here for additional data file.


Halyomorpha halys, is an invasive hemipteran that causes significant economic losses to various agricultural products around the world. Recently, the pyrokinin and capa genes that express multiple neuropeptides were described in this species. Here we report six pyrokinin and capa GPCRs including two splice variants, and evaluate their (a) ability to respond to neuropeptides in cell-based assays, and (b) expression levels by RT-PCR. Functional studies revealed that the H. halys pyrokinin receptor-1  responded to the pyrokinin 2 (PK2) type peptide. RT-PCR results revealed that these receptors had little or no expression in the tissues tested, including the whole body, central nervous system, midgut, Malpighian tubules, and reproductive organs of males and females. HalhaPK-R2 showed the strongest response to PK2 peptides and a moderate response to pyrokinin 1 (PK1) type peptides , and was expressed in all tissues tested. HalhaPK-R3a & b responded to both PK1 and PK2 peptides. Their gene expression was restricted mostly to the central nervous system and Malpighian tubules. All PK receptors were dominantly expressed in the fifth nymph. HalhaCAPA-R responded specifically to CAPA-PVK peptides (PVK1 and PVK2), and was highly expressed in the Malpighian tubules with low to moderate expression in other tissues, and life stages. Of the six GPCRs, HalhaPK-R3b showed the strongest response to PK1. Our experiments associated the following peptide ligands to the six GPCRs: HalhaPK-R1a & b and HalhaPK-R2 are activated by PK2 peptides, HalhaPK-R3a & b are activated by PK1 (= DH) peptides, and HalhaCAPA-R is activated by PVK peptides. These results pave the way for investigations into the biological functions of H. halys PK and CAPA peptides, and possible species-specific management of H. halys.The brown marmorated stink bug,  They regulate or modulate a variety of physiological actions, such as fat body homeostasis, feeding, digestion, excretion, circulation, reproduction, metamorphosis, and behavior throughout all life stages . The PRX Insecta . The pepDrosophila genome identified a variety of GPCRs for PRXamide peptides that are orthologous to the vertebrate neuromedin U receptor (NmU-R) (Drosophila PRXamide peptides were first identified and found to be related to the vertebrate peptide neuromedin U (NmU) (Helicoverpa zea (Bombyx mori (The neuropeptides activate specific G protein-coupled receptors (GPCRs) that are involved in essential biological processes. Data mining and computational analyses of the  (NmU-R) . The hyp U (NmU) . Since terpa zea , followebyx mori . To datebyx mori .Nezara viridula , but no PK1 (= DH) peptide; and the capa gene encoding two periviscerokinin (PVK) peptides (CAPA-PVK1 and CAPA-PVK2) and a CAPA-DH (= PK1-like) peptide  using random hexamers and oligo-dT primers as described previously  and gene-specific primers to amplify full-length sequences using the following PCR conditions: an initial 8 min incubation at 94°C followed by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 90 s. Three positive colonies were each inoculated into LB media and grown overnight. Plasmids were purified from bacterial cultures using the GeneJet Plasmid Miniprep Kit (Thermo Fisher Scientific) and Sanger sequenced to confirm the correct sequence and orientation of each gene within the pIB vectors. One plasmid containing each HalhaPK/CAPA-R gene with the correct sequence and orientation was chosen for transfection of insect cell lines.Total RNA was isolated from various tissues of male and female eviously . Primerseviously  were desFive micrograms of each purified pIB/HalhaPK-R (or CAPA-R) plasmid and 15 μL of Cellfectin II reagent (Thermo Fisher Scientific) were diluted into 500 μL of serum free Insectagro Sf9 medium  and allowed to incubate for 10 min at room temperature, after which they were mixed together and allowed to incubate for an additional 15–20 min. Each pIB/HalhaPK-R (or CAPA-R) Cellfectin mixture was then added to T-25 cell culture flasks containing Sf9 cells at 50% confluency and incubated overnight at 28°C. The following morning the media was removed from each flask and replaced with media containing 20 μg/mL blasticidin (Corning). Each transfected cell line was cultured in the presence of 20 μg/mL blasticidin for ∼3 weeks until a blasticidin-resistant cell line was established, after which the blasticidin concentration was reduced to 10 μg/mL and the cell lines were frozen prior to testing in functional assays.H. halys pyrokinin and capa genes used in this study have been recently identified . The plated cells were then incubated at 28°C for 48 h, after which the media was removed from the plates and the wells were rinsed with serum-free media, then loaded with 95 μL of 1X FLIPR Calcium 6 assay reagent . Plates were incubated at room temperature for 60 min then transferred to a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) to measure cell fluorescence intensity from intracellular calcium influx after peptide-induced receptor activation. Wells of cells were then treated with 5 μL of water or 20X concentrations of each peptide using the automated injection system of the FlexStation plate reader. In all experiments, fluorescence was measured every 5 s for 30 s prior to treatment and for 2 min after exposure. In all cases, the maximum response obtained occurred ∼20 s after exposure to ligand. The mean response of four wells receiving the same treatment was calculated and expressed as a change in fluorescence from baseline (prior to treatment) to maximum response. For initial screening, each cell line was exposed to a single concentration of 500 nM of each peptide or control. Compounds that elicited responses were tested further in concentration-response experiments. Concentration-response curves and EC50 values were generated using the variable slope, four parameter non-linear regression function of GraphPad Prism data analysis . Each cell line was tested for its response to peptides in at least three independent experiments.For each cell line expressing a specific The following tissues: central nervous system, midgut, Malpighian tubules, testis and ovary, were dissected from 5 to 10 days-old adults and the total RNA was extracted as described previously . cDNA waTM Real-Time PCR System (Applied Biosystems). Amplification specificity was followed by melting curve analysis over a 60–95°C range. Gene expression levels for the developmental stages were compared using rpn2 as a reference gene.Samples were collected from the following developmental stages: eggs, 1st to 5th nymphs, adult males and adult females . Total RNA isolation, cDNA synthesis, and quantitative real-time PCR (qRT-PCR) were performed as described previously . BrieflyH. halys . These were designated as HalhaPK-R1a, HalhaPK-R1b, HalhaPK-R2, HalhaPK-R3a, HalhaPK-R3b, and one, HalhaCAPA-R, as a CAPA receptor. Sequences of the six receptors were aligned and their transmembrane domains determined  and one missing exon 6 . In addition, there were 6 non-synonymous mutations between the two versions of the proteins, which were validated by independent PCR methods and their presence in the genome. In the genome, some of the exons code for the HalhaPK-R1a transcripts and some code for HalhaPK-R1b transcripts, indicating that the genome sequence is an amalgamation of different HalhaPK-R1 alleles that were present in the pooled DNA used to generate the genome. HalhaPK-R2 was comprised of 5 exons spread across a 99,734 bp region of scaffold629, and was found as a complete transcript in contigs XM_014427306.1 and XM_014427307.1 in the i5k database. HalhaPK-R2 was amplified via PCR and the sequence of the resulting product exactly matched that of the transcriptome and genome. HalhaPK-R3a and HalhaPK-R3b were both present in the i5k genome and were comprised of 5 exons spread across a 56,025 bp region of scaffold1013, with two alternatives of exon 5. Both HalhaPK-R3a and HalhaPK-R3b were present in the i5k transcriptome on contigs XM_014431884.1 and XM_014431883.1, respectively. HalhaCAPA-R was present in the i5k genomic database, comprised of 4 exons spread across scaffold1099  and scaffold3190 (exon 3), and in the transcriptome spread across two un-assembled contigs (XM_014432674.1 and XM_014437682.1). The HalhaCAPA-R transcriptomic and genomic sequences were identical.H. halys peptides or a negative vehicle control , with varying sensitivities and degrees of absolute magnitude . HalhaCAH. halys GPCRs were differentially expressed in male and female adult tissues  were expressed the most in the last nymphal stage (N5), whereas the CAPA receptor gene  showed broad-range expression throughout the life cycle, except in the egg stage . Among dH. halys genomic and transcriptomic databases. The GPCRs were functionally characterized using PK and CAPA peptides in vitro. Upon amplification of these receptors via PCR it was discovered that two PK receptors, HalhaPK-R1 and HalhaPK-R3, were transcribed as two alternatively-spliced variants, which is not unusual among PBAN/PK receptors in moths (R. prolixus (G protein-coupled receptors (GPCRs) are readily identified from insect genome sequences based on the presence of a well conserved, seven-transmembrane-domain topography . We idenin moths . The “a”in moths . We did prolixus .H. halys PK and CAPA receptors are expected to respond to PK peptides encoded from the PBAN/pyrokinin gene, and CAPA-PVK peptides encoded from the capa gene, respectively. In this study, HalhaCAPA-R was clearly distinguished from the other five receptors. It responded strongly only to CAPA-PVK peptides containing the PRVamide conserved motifs in the C-termini, but had no response to PK type peptides containing the PRLamide conserved motifs in the C-termini. Also, CAPA-PVK peptides did not activate any of the other PK receptors tested in a concentration-dependent manner. The CAPA receptor was expressed in all tissues and life stages we tested in this study. In addition, the CAPA receptor expression levels were much higher than the other H. halys receptors. In fact, capa genes are ubiquitously found across invertebrate phyla including Arthropoda  or PK2 (= PBAN) receptors. Although HalhaPK-Rs 1a/b, 2, 3a/b all responded to PK2 peptides, HalhaPK-R1a/b responded weakly to PK2 peptides and had very low expression in the body, suggesting they might be losing their biological relevance in Helicoverpa zea  or only CAPA peptides, respectively. Actually, these two receptors are the most dominant in the H. halys organs tested in this study. Although HalhaPK-R2 is expected to be the primary receptor for PK2 peptides, HalhaPK-R3b strongly responded to both PK1 and PK2 peptides, thus it may be an alternative receptor in H. halys. Previously, it was found that PK2 receptors are more promiscuous in other insects, responding to both PK2 and DH ligands, but insect DH receptors tend to respond specifically to DH type peptides (H. halys have evolved differently than in other previously studied insect groups.From the first report of a PBAN receptor in erpa zea  to the rrnacalis , many GPreported . Interespeptides . Other speptides , b. It iH. halys PK GPCRs in selected tissues of H. halys and compared them between male and female. RT-PCR results revealed that the expression patterns of H. halys receptors were similar between male and female, with one exception being that HalhaPK-R2 was relatively more abundant in the Malpighian tubules and central nervous system of males than in female adults (H. halys tissues. Interestingly, strong transcription of HalhaPK-R2 was detected in testis and ovary, indicating a potential function involving muscle contraction or nutritional metabolism in these reproductive organs. PK2 peptides were previously isolated from cockroaches, and demonstrated to stimulate the contraction of hindgut muscles (H. halys.We then examined the expression patterns of the mRNAs of four e adults . Like th muscles . Our RT-pyrokinin and capa genes in the brown marmorated stink bug. These receptors might have pivotal roles in the regulation of critical physiological processes that govern the development and survival of this insect. Now that these receptors have been identified and characterized, they can serve as biological targets in the development of H. halys specific pest control methods.In this study, we identified six GPCRs that show varying response profiles to peptides produced from The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.S-JA, JC, and MC performed all the experiments and analyzed the data. RV and the other authors wrote and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.